The maintenance of DNA replication fork stability under conditions of DNA damage and at natural replication pause sites is essential for genome stability. Here, we describe a novel role for the F-box protein Dia2 in promoting genome stability in the budding yeast Saccharomyces cerevisiae. Like most other F-box proteins, Dia2 forms a Skp1-Cdc53/Cullin-F-box (SCF) E3 ubiquitin-ligase complex. Systematic analysis of genetic interactions between dia2D and 4400 viable gene deletion mutants revealed synthetic lethal/ synthetic sick interactions with a broad spectrum of DNA replication, recombination, checkpoint, and chromatin-remodeling pathways. dia2D strains exhibit constitutive activation of the checkpoint kinase Rad53 and elevated counts of endogenous DNA repair foci and are unable to overcome MMS-induced replicative stress. Notably, dia2D strains display a high rate of gross chromosomal rearrangements (GCRs) that involve the rDNA locus and an increase in extrachromosomal rDNA circle (ERC) formation, consistent with an observed enrichment of Dia2 in the nucleolus. These results suggest that Dia2 is essential for stable passage of replication forks through regions of damaged DNA and natural fragile regions, particularly the replication fork barrier (RFB) of rDNA repeat loci. We propose that the SCF Dia2 ubiquitin ligase serves to modify or degrade protein substrates that would otherwise impede the replication fork in problematic regions of the genome. Collapse of replication forks results in the formation of DNA double-strand breaks (DSBs) that can then lead to illegitimate recombination and genome rearrangements, both of which are thought to be underlying causes of many human cancers (Lengauer et al. 1998). Physical impediments to replication fork progression include tightly bound non-nucleosomal protein-DNA complexes, DNA secondary structures, and regions of DNA damage, whereas inadequate dNTP pools cause forks to slow and eventually stall in a non-locus-specific manner (Branzei and Foiani 2005). Accumulated DNA damage or stalled replication forks elicit a checkpoint response that results in a delay of the cell cycle, induction of damage responsive genes, and the repair or bypass of the DNA lesion (Melo and Toczyski 2002;Branzei and Foiani 2005). The DNA damage checkpoint is activated upon detection of DNA lesions in G 1 and G 2 phase, and in S-phase the latter sometimes is referred to as the intra-S checkpoint. A second response in S-phase, referred to as the replication checkpoint, is the response to delayed DNA synthesis as caused by lowered dNTP pools upon inhibition of ribonucleotide reductase by hydroxyurea (HU). It is likely that the replication and intra-S checkpoint pathways are integrated such that the key signal is stalled or slowed replication forks, due to either dNTP shortage or collision with DNA damage. The only essential function of the S-phase checkpoint is to stabilize the fork when cells undergo replicative stress (Terceroet al. 2003) and thereby prevent the accumulation of recombinog...

The ability to quantitatively compare protein levels across different regions of the brain to identify disease mechanisms remains a fundamental research challenge. It requires both a robust method to efficiently isolate proteins from small amounts of tissue and a differential technique that provides a sensitive and comprehensive analysis of these proteins. Here, we describe a proteomic approach for the quantitative mapping of membrane proteins between mouse fore- and hindbrain regions. The approach focuses primarily on a recently developed method for the fractionation of membranes and on-membrane protein digestion, but incorporates off-line SCX-fractionation of the peptide mixture and nano-LC-MS/MS analysis using an LTQ-FT-ICR instrument as part of the analytical method. Comparison of mass spectral peak intensities between samples, mapping of peaks to peptides and protein sequences, and statistical analysis were performed using in-house differential analysis software (DAS). In total, 1213 proteins were identified and 967 were quantified; 81% of the identified proteins were known membrane proteins and 38% of the protein sequences were predicted to contain transmembrane helices. Although this paper focuses primarily on characterizing the efficiency of this purification method from a typical sample set, for many of the quantified proteins such as glutamate receptors, GABA receptors, calcium channel subunits, and ATPases, the observed ratios of protein abundance were in good agreement with the known mRNA expression levels and/or intensities of immunostaining in rostral and caudal regions of murine brain. This suggests that the approach would be well-suited for incorporation in more rigorous, larger scale quantitative analysis designed to achieve biological significance.

Metastasis leads to the death of most cancer patients, and basal breast cancer is the most aggressive breast tumor type. Metastasis involves a complex cell migration process dependent on cytoskeletal remodeling such that targeting such remodeling in tumor cells could be clinically beneficial. Here we show that Hormonally Upregulated Neu-associated Kinase (HUNK) is dramatically downregulated in tumor samples and cell lines derived from basal breast cancers. Reconstitution of HUNK expression in basal breast cancer cell lines blocked actin polymerization and reduced cell motility, resulting in decreased metastases in two in vivo murine cancer models. Mechanistically, HUNK overexpression sustained the constitutive phosphorylation and inactivation of cofilin-1 (CFL-1), thereby blocking the incorporation of new actin monomers into actin filaments. HUNK reconstitution in basal breast cancer cell lines prevented protein phosphatase 2-A (PP2A), a phosphatase putatively acting on CFL-1, from binding to CFL-1. Our investigation of HUNK suggests that the interaction between PP2A and CFL-1 may be a target for antimetastasis therapy, particularly for basal breast cancers.M etastasis is a hallmark of cancer and remains the major cause of cancer-related mortality (90%) (1). Currently, there is no FDA-approved drug that specifically blocks metastasis. Metastasis is a multistep process that requires a cancer cell to leave a primary tumor, intravasate, survive in the blood, extravasate, migrate, invade through basement membranes and connective tissues, and establish a viable tumor in a distant site (2). Cytoskeletal reorganization and cell movement underlie all these events (3), and disruption of these processes could therefore constitute an effective anticancer approach.The major subtypes of breast cancer (luminal A/B/C, HER-2, and basal) can be distinguished by their gene expression profiles (4). The basal subtype is the most aggressive, has the worst prognosis, and shows the greatest extent of metastasis (4, 5). To identify molecules whose expression varies by breast cancer subtype and might be linked to metastasis, we screened the online data of Sorlie and colleagues (4) for candidate promoters and suppressors of metastasis. Our hypothesis was that the mRNA expression of kinases involved in cell migration and invasion should be altered in basal breast cancers relative to the mRNA profiles of other breast cancer subtypes. One molecule emerging from this screen was Hormonally Up-regulated Neuassociated Kinase (HUNK), an 80-kDa protein (6). HUNK was down-regulated almost threefold in basal cancer samples compared to the other subtypes. HUNK contains a 260-aa domain predicted to have serine/threonine kinase activity; however, to date, no clear kinase activity, substrates, interacting proteins, or indeed physiological role for HUNK have been identified (6, 7). In normal murine mammary cells, HUNK levels vary with the hormonal cycle (6). With respect to transformed cells, Wertheim et al. recently reported that HUNK is highly express...